Recent studies on the association of hemoglobin with isolated membranes from human erythrocytes have shown there to be a hemoglobin binding site on the cytoplasmic surface of the plasma membrane. The specific membrane protein(s) responsible for this binding and the question of similarity of binding of hemoglobin A and S to this site will be established by further cross-linking studies as outlined in the proposal. Manipulation of the arrangement of membrane proteins in isolated ghosts by solvents and detergents and the consequent impact on the binding of hemoglobin will also aid in determining the site(s) of hemoglobin binding. Measurements of resonance energy transfer between a fluorescent probe inserted into the lipid bilayer and the heme of membrane-bound hemoglobin are in progress and will provide values for the affinity and number of molecules of oxy and deoxy hemoglobin A and S associated with the membrane. It has been suggested that the binding of certain agents to the outer surface of erythrocyte ghosts alters the binding of hemoglobin to the cytoplasmic surface of the membrane. These experiments will be repeated in whole erythrocytes from persons with Sickle Cell Anemia and any changes in the rates of sickle cell formation will be monitored by rapid-mixing experiments. Thus, the possible role of the binding of hemoglobin to the erythrocyte plasma membrane in Sickle Cell Disease will be assessed for the first time.